The present invention relates to the field of determining fungal biomass in a variety of samples including environmental samples, food products, plant materials, building construction materials, human and animal body samples and industrial fungal cultures.
Fungal species are used in the fermentation of foods and for the production of desired gene products like enzymes and antibiotics. Fungi may also cause spoilage of foods and agricultural products, cause rot in building materials, and may be pathogenic to mammals including humans.
Currently used methods for detecting fungal biomass include extracting fungus specific cell components such as ergosterol (Grant and West, 1986) and phospholipid fatty acids (PLFA) (Frosteg{dot over (a)}rd et al., 1993), selectively staining cellular organelles (Kropp, 1990; U.S. Pat. No. 5,445,946), fluorescently detecting fungal metabolites present in cellular extracts (SU 1,744,114; JP 7,095,898), and immunological detection of structural components of the fungal cell wall (U.S. Pat. No. 5,004,699).
However, all of these methods involve several drawbacks, in particular due to complicated sample preparations including disruption of cells and hyphae and isolation of cell components. In addition, the methods generally involve extended assay periods and besides, they are time consuming, laborious and require specialised technical skills.
There is therefore a need for an improved, simple and rapid method of selectively determining fungal biomass which can be performed without the above drawbacks.
It has now been found that a reproducible correlation exists between certain enzymatic activities which is present in substantially all fungal species, and the amount of fungal biomass and/or the amount of fungal cell components. This discovery has led to the development of a novel method of determining fungal biomass that fulfills the above need for an improved method.
Accordingly, the present invention pertains in one aspect to a method of selectively detecting a fungal biomass in a sample, comprising detecting the amount, presence or activity of at least one enzyme that is present in substantially all fungal species, the amount or activity of which enzyme is correlated with the amount of fungal biomass present in the sample, the detection being made under conditions where enzymes of non-fungal origin, if present, cannot be detected. In particular, there is provided a method comprising the steps of (i) contacting a sample with a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of a selectively detectable fungal enzymatic activity, and (ii) detecting the released moiety.
In a further aspect, the invention pertains to a product comprising (i) an agent that reacts with an enzyme that is present in substantially all fungal species and the amount or activity of which enzyme is correlated with the amount of fungal biomass present in a sample, the reaction between the enzyme and the agent resulting in a detectable signal and (ii) means for detecting said signal, as a combined system for the detection of a fungal biomass present in the sample.
The invention provides, as it mentioned above, a method of selectively detecting a fungal biomass in a sample, comprising detecting the amount, presence or activity of at least one enzyme being present in substantially all fungal species and the amount or activity of which is correlated with the amount of fungal biomass present in the sample.
As used herein, the expression xe2x80x9cselectively detectingxe2x80x9d indicates that, when a sample is tested in accordance with the invention, the test conditions are selected so as to exclude any detection of non-fungal enzymes or enzymatic activities which could otherwise interfere with a selective assay for fungal enzymes.
In the present context, the expression xe2x80x9cfungal biomassxe2x80x9d refers to any cellular components of fungal species as they are defined in Henderson""s Dictionary of Biological Terms, 10th edition, Longman Scientific and Technical, 1990. Thus, as used herein fungal biomass includes single cells, mycelia, thalli, hyphae and spores of the fungal species mentioned in the above reference book. Fungal species as defined herein include species belonging to the subdivisions Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina.
In one embodiment, the method of the invention is based on the detection in a sample to be tested of the activity of at least one fungal enzyme by contacting the sample with a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of the fungal enzyme followed by detecting the released moiety.
Thus, an assay for detecting a fungal enzymatic activity can be based upon specific cleavage of a substrate molecule into one or more readily detectable moieties. Fluorogenic moieties can be detected with a high sensitivity and the use of substrate molecules comprising fluorescently detectable moieties in conventional assays of enzymatic activities is well characterised and can be used in accordance with the invention. As an example, the high sensitivity of detection of fluorogenic moieties has facilitated the development of assays for the detection of e.g. chitinase produced by bacterial species (McCreath and Gooday, 1992) and xcex2-glucanase activities in fungal species, including yeast (Claeyssens et al., 1989)
Thus, the present method can include the detection of an enzymatic activity that is associated with the metabolism of cell structural components. As used in this context, the expression xe2x80x9cstructural componentsxe2x80x9d includes naturally occurring substances which confer rigidity and mechanical strength to fungal cell walls including septae such as e.g. chitin, chitosan, cellulose, glycogen, glucan, polygalactosamine and polypeptides.
Fungal enzymatic activities which can be used in the present method include those naturally produced in a fungal biomass to be detected. Alternatively, a detectable fungal enzymatic activity in a given fungal biomass can be expressed from a gene inserted by genetic recombination.
In accordance with the invention, detectable enzymatic activities are preferably activities that are expressed constitutively, expressed in all growth phases of the fungal biomass and/or expressed independently of the physiological state of the fungal biomass. The enzymatic activities can be cell associated and/or extracellular.
In other embodiments, the method is based on a detectable enzymatic activity which is expressed in both the presence and absence of biologically cleavable polymers present in a fungal cell wall, such as e.g. chitin, glucans and polypeptides. In yet another embodiment, the detectable enzymatic activity is expressed in both the presence and absence of biologically cleavable polymers such as polysaccharides e.g. including cellulose, hemicellulose, amylose, amylopectin, mannan, xanthan, xylan, arabinan and galactan or is a fungal enzymatic activity that degrades any of such polysaccharides or polypeptides. A presently preferred embodiment of the method according to the invention is based on the detection in a sample of an enzymatic activity selected from an enzyme hydrolysing xcex2-(1-4) bonds between N-acetyl-hexosaminide groups and an enzyme synthesising such bonds. In this connection, xcex2-(1-4) bond hydrolysing enzymes include any fungal enzymatic activity that hydrolyses such bonds in polymers and glycosylated proteins containing N-acetylglucosaminide groups, such as the polysaccharides chitin and/or chitosan which are present in substantially all fungal species.
Accordingly, the present method can be based upon detection of a fungal enzymatic activity associated with chitin metabolism, in particular a chitinase (E.C. 3.2.1.14), a xcex2-N-acetylhexosaminidase (E.C. 3.2.1.52) and/or a chitin synthase (E.C. 2.4.1.16). Alternatively, the method is based on an enzymatic activity that is associated with the metabolism of chitosan, i.e. an at least partially deacetylated chitin. An example of such an enzyme activity is chitosanase (E.C. 3.2.1.132) (The above E.C. numbers refer to Enzyme Data Bank, Release 21.0/October 1996).
Besides being based on detection of the activity of a fungal biomass associated enzymatic activity, the present method encompasses any other assay procedure permitting the detection of a fungal enzyme, the amount of which is correlated with the fungal biomass. Such procedures include as examples detecting the amount of the fungal enzyme immunologically and the detection of DNA and/or RNA sequences coding for the enzymatic activity of interest. Such procedures can be based on methods which are well-known in the art and include e.g. the use of antibodies, optionally labelled with detectable moieties and the use of oligonucleotide probes that hybridizes selectively to the DNA or RNA sequences.
It is a significant aspect of the present invention that a wide variety of fungal species, when tested for their ability to enzymatically cleave substrates labelled with fluorogenic moieties, produce an enzymatic activity releasing fluorescently detectable moieties, the amount of which is correlated with the amount of ergosterol and fungus related phospholipid fatty acids (PLFA), the presence of which are currently used in the determination of a fungal biomass.
Surprisingly, the amount of the released fluorescently detectable moieties is directly correlated with the biomass of the assayed fungal species producing the above enzymatic activity. This correlation is indicative of a constitutive expression of the substrate cleaving enzymatic activity.
In another preferred embodiment the method comprises a further step of correlating the activity or the amount of fungal enzyme with a fungal biomass parameter of the fungal biomass such as e.g. the amount of fungal biomass present in the sample, as determined by directly measuring the weight of the fungal biomass. The fungal biomass parameter can also be a metabolite, an additional enzymatic activity or an indicator of the physiological state of said fungal biomass. In this context, suitable fungal metabolites include fatty acids, polysaccharides and parts hereof, polyketides, steroids, shikimic acids, alkaloids, a pigment naturally produced by a fungal species such as e.g. astaxanthin, carotenoids, riboflavins, terpenoids and derivatives hereof, antibiotics such as e.g. penicillins.
In a particular embodiment of the method according to the invention, a detectable moiety released from a substrate for the fungal enzyme is correlated with the amount of fungal biomass present in the sample as determined directly by measuring the weight of the fungal biomass. Preferably the weight is the dry weight of the fungal biomass. Having established such a correlation in the form of e.g. a standard curve, the method can be used to establish further suitable standard curves e.g. illustrating, under appropriate experimental and/or industrially relevant conditions, the relationship between the amount of released moiety and the amount of the fungal biomass. Standard curves may be produced for a number of experimental and/or industrially relevant conditions so as to provide a set of physiological parameters associated with the physiological state of a fungal species under a variety of conditions. Such parameters include growth rate, growth phase, temperature, pH, oxygen content, growth medium composition including the presence/absence of preferably metabolisable nutrients, osmotic strength, and the presence or absence of essential cell constituents or precursors herefor.
The indicator of the physiological state of said fungal biomass shall also be understood to include the presence or absence of certain metabolites or polypeptides produced i) in certain growth phases, ii) in response to changing environ mental conditions, or iii) in response to the presence or absence of cellular or chemical components.
The correlation between the released detectable moiety and an indicator of the physiological state of the fungal biomass can also be applied to establish a reliable method for assessing xe2x80x9cphysiological fitness and adaptabilityxe2x80x9d of a fungal biomass. This is particularly important in the detection of a fungal biomass which is genetically engineered, selected or otherwise improved for a particular purpose, typically an increased production of a metabolite, an antibiotic or a desired gene product. Thus, the production in large amounts of homologous and/or heterologous polypeptides may lead to misfolding of the produced polypeptide and/or overloading of the cellular transport or secretion apparatus. These undesirable phenomena are likely to induce a stress response which, when present in a xe2x80x9csemi-permanent statexe2x80x9d, is likely to reduce the production yield. In accordance with the present method, a detectable moiety that is released by the fungal enzymatic activity can be detected by any spectroscopic method. Thus, suitable spectroscopic methods include methods for determining a selective absorption of electromagnetic radiation by substrate molecules and moieties released herefrom such as fluorometry, wherein the fluorescence emitted by the released moiety is determined. A selective detection of a released and fluorescently detectable moiety can also be achieved by using a filter absorbing light of all but desired, predetermined wavelengths, which pass through the filter prior to being detected.
When it is desired to detect more than one substrate moiety, the different moieties should emit fluorescence which is detected at different wavelengths and/or by using suitable filters as described above.
Alternative methods of detecting selective absorption of electromagnetic radiation include ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, including pulsed Fourier transform NMR, mass spectrometry, X-ray diffraction, microwave absorption, electron spin resonance, optical rotatory dispersion and circular dichroism.
Ultraviolet spectroscopy is used to detect conjugated systems, because the promotion of electrons from the ground state to the excited state of such systems gives rise to absorption in this region of the spectrum. Infrared spectroscopy is used to detect and identify the vibrations of molecules and in particular the characteristic vibrations of the double and triple bonds present in many functional groups.
Nuclear magnetic resonance spectroscopy uses a longer wavelength of the electromagnetic spectrum to detect changes in the alignment of nuclear magnets in strong magnetic fields. The precise frequency of absorption is a very sensitive measure of the magnetic, and hence the chemical, environment of such nuclei. Moreover, the number and disposition of neighbouring magnetic nuclei influence the appearance of that absorption in a well-defined way. The result is a considerable information about the arrangement of functional groups and hydrocarbon residues in e.g. a moiety part of or released from the substrate molecule.
Mass spectrometry measures the mass-to-charge ratio of substrate moieties which have been charged by electron bombardment. Structural information comes from the moderately predictable fragmentation of substrate molecules, including a correlation of charged moieties with likely structures. X-ray diffraction can be used to identify centres of high electron density, such as e.g. atoms. Microwave absorption is used to measure molecular rotations of substrate moieties.
Electron spin resonance detects unpaired electrons and can be used to measure the distribution of electron densities in substrate moieties such as e.g. released radicals. Optical rotatory dispersion and circular dichroism use visible and ultraviolet light for determining the correlation of changes in rotatory energy of substrate moieties with changes of polarized light; such measurements can often be related to the absolute configuration of molecules.
Thus, it will be understood that moieties are xe2x80x9cselectivelyxe2x80x9d detectable by application of results obtainable from one or more of the above-mentioned methods, such as e.g. UV maxima, IR frequencies, NMR chemical shifts and coupling constants, and common mass fragments found in mass spectra.
The method of the invention is suitable for the detection of fungal biomass including species belonging to Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina, or a mixture hereof. In one embodiment the invention pertains to the detection of fungal biomass comprising viable propagules in the form of mycelia, conidia, spores and single cells, such as yeast. In another embodiment of the invention, the fungal biomass in the assayed sample comprises viable fungal biomass and/or non-viable fungal biomass.
The method can be used to detect fungal biomass in small amounts such as an amount which is at the most 1 xcexcg, preferably at the most 0.1 xcexcg, more preferably at the most 0.01 xcexcg and most preferably at the most 0.001 xcexcg is detectable.
In one embodiment, the method according to the present invention comprises the further step of pre-incubating the sample in a suitable medium supporting the growth of fungal biomass, prior to the actual assaying for the presence hereof. The objective of the pre-incubation is to allow any fungal biomass to propagate and thus contribute to an increased sensitivity of the assaying procedure described herein below. The pre-incubation can include adding a compound inhibiting non-fungal biological activity to a suitable growth medium. Compounds that are useful for inhibiting non-fungal biological activities include antibiotics, such as e.g. chloramphenicol and/or streptomycin, bacteriocins and chemicals having selective growth inhibiting effects on bacteria, such as e.g. sulphites or metal ions. The length of the pre-incubation period depends on the amount of fungal biomass and the amount of non-fungal biomass present in the sample.
In accordance with the present method, a sample can be pre-incubated several times under conditions selectively suppressing pressing growth of fungal biomass or non-fungal biomass. Such pre-incubations can precede a series of assaying procedures wherein i) the total biomass is determined quantitatively (i.e. no suppression of growth), ii) fungal biomass is determined quantitatively (i.e. suppression of growth of non-fungal biomass), and iii) non-fungal biomass is determined quantitatively (i.e. suppression of growth of fungal biomass).
The present method may include the addition of an inducer substance capable of inducing or enhancing the detectable fungal enzymatic activity. The inducer can be added i) during the pre-incubation period as mentioned above, ii) immediately prior to the assay procedure or iii) at suitable time intervals during the assay procedure. When it is preferred to determine the presence of non-fungal biomass, the addition of an inducer substance capable of inducing or enhancing the non-fungal enzymatic activity can be added.
Useful inducers include monomers and/or oligomers, such as e.g. dimers and trimers, of substances making up polysaccharides such as e.g. cellulose, hemicellulose, amylose, amylopectin, chitin, mannan, xanthan, xylan, arabinan and galactan. Particularly useful inducers are glucosamines, such as e.g. N-acetyl-glucosamine and amino-substituted derivatives hereof.
In yet another embodiment, the method according to the invention comprises, following the assay procedure, a further step of incubating the sample in a suitable selective medium permitting growth of only i) fungal biomass or ii) non-fungal biomass. Growth of non-fungal biomass, such as e.g. bacteria, may thus lead to determining bacterial species comprised in the sample by using state of the art techniques. Similarly, the fungal biomass comprised in the sample may be further differentiated or identified by growth on selective media supplemented with e.g. a particular carbon source, which supports growth and/or the development of phenotype traits of one or more fungal species e.g. belonging to Zygomycotina, Ascomycotina, Basidiomycotina or Deuteromycotina.
In the detection of enzymatic activity in accordance with the invention, useful substrate molecules comprising fluorescently and/or chromogenically detectable moieties are particularly preferred such as substrate molecules including a fluorogenic and/or chromogenic moiety such as a 4-methylumbelliferone moiety or derivatives hereof, e.g. 4-methylumbelliferyl-N-acetyl-xcex2-D-glucosaminide (4-MU-GlcNAc). It has been found that substantially all fungal species are capable of constitutively cleaving this substrate whereas none of a range of bacterial species tested had this capability.
Alternative substrate molecules include 5-bromo-6-chloro-3-indolyl-2-acetamido-2-deoxy-xcex2-D-gluco-pyranoside, 5-bromo-4-chloro-3-indolyl-N-acetyl-xcex2-D-glucosaminide, indolyl-2-acetamido-2-deoxy-xcex2-D-gluco-pyranoside, 4-nitrophenyl-N-acetyl-xcex2-D-glucosaminide, xcex2-trifluoromethylumbelliferyl-N-acetyl-xcex2-D-glucosaminide, N-methylum-indolyl-N-acetyl-xcex2-D-glucosaminide, 5-iodo-3-indolyl-N-acetyl-xcex2-D-glucosaminide, 4-methylumbelliferyl-xcex2-D-N,Nxe2x80x2,Nxe2x80x3-triacetylchitotriose, 4-methylumbelliferyl-xcex2-D-N,Nxe2x80x2-diacetylchitobioside, 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-xcex2-D-glucosaminide, 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy-xcex2-D-glucopyronoside), 4-methylumbelliferyl-N-acetyl-xcex1-D-glucosaminide, 4-methylumbelliferyl-N-acetylgalactosaminide, resorufin-N-acetyl-xcex2-D-glucosaminide, 4-methylumbelliferyl-N-acetyl-xcex1-D-glucosaminide and DDAO (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)N-acetyl-xcex2-D-glucosaminide) and all N-actyl-xcex2-D-glucosaminide oligomer derivatives of DDAO.
In preferred embodiments of the invention, the released fluorometrically and/or chromogenically detectable moiety is detectable in an amount of at the most 100 picomoles, preferably at the most 50 picomoles, more preferably at the most 20 picomoles, even more preferably at the most 10 picomoles and most preferably, the released moiety is detectable in an amount of at the most 1 picomole.
Although preferred embodiments of the invention pertain to a fluorometric and/or chromogenic detection of the released moiety, the method includes an embodiment wherein the released moiety is visibly detectable e.g. by luminescence without any further quantification or data processing.
The invention also provides a method comprising the further step of using at least one additional substrate molecule comprising a releasable moiety which is detectable. This moiety may be released either by a fungal enzymatic activity, a general microbial enzymatic activity or a non-fungal enzymatic activity.
The detectable moiety comprised in the additional substrate molecule may thus serve to differentiate between, or further facilitate identification of, i) one or more fungal species comprised in the sample, ii) microbial biomass comprised in the sample and iii) non-fungal biomass comprised in the sample.
Use of the method according to this particular embodiment would thus facilitate a selective detection in the sample of interest of microbial biomass, fungal biomass and non-fungal biomass, respectively. This is particularly relevant when the sample to be assayed is obtained from an environment also permitting growth of e.g. non-fungal biomass.
Although primarily providing the means of determining a fungal biomass, the method may also in particular embodiments be used in the determination of a particular fungal species, a particular microbial biomass or a non-fungal biomass. Thus, it is well known that chitin is a major polysaccharide of the cell wall of fungi. Chitin cleaving enzymes facilitating incorporation of the monomeric elements of chitin, N-acetyl-glucosamine, into the fungal cell wall may thus be considered candidates for selectively detectable fungal enzymatic activities.
By analogy, the present invention provides, in one contemplatable embodiment, a method of determining e.g. a particular fungal species, a particular microbial biomass or non-fungal biomass based on the presence of selectively detectable enzymatic activities either cleaving or synthesising polysaccharides.
As one example of such a contemplatable embodiment, the fungal species Penicillium charlesii produces a furanose linked polysaccharide comprising galactan, when grown on glucose whereas, in the absence of glucose or during starvation, this polysaccharide is believed to be cleaved by a galactan cleaving enzymatic activity present in Penicillium charlesii. Detection of this enzymatic activity can be used to selectively detect Penicillium charlesii. 
In terms of determining bacterial biomass, reference can be made to dextran synthesising strains of Leuconostoc growing on sucrose. Enzymatic activities capable of synthesising dextran may thus form the basis of determining Leuconostoc biomass. In yet another example, the xanthan synthesising activities of Xanthomonas campestris could form the basis of a method of determining this bacterial species.
In a further example, the bacterial polysaccharide curdlan is produced by Alcaligenes faecalis and the detection of enzymatic activities involved in this production may be used to selectively determine Alcaligenes faecalis biomass.
In accordance with the invention, the time period of contacting the sample with the substrate molecule is at the most 24 hours, preferably at the most 6 hours, such as at the most 3 hours, more preferably at the most 30 minutes, even more preferably at the most 10 minutes such as at the most 1 minute and most preferably, the time of contacting the sample with the substrate molecule is at the most 30 seconds. The samples can be assayed in micro-titre plates.
The method of the invention may include collection of samples and/or detection of the released moiety at suitable time points over a suitable time interval. There is also provided a method according to the invention wherein the released moiety is detected continuously e.g. by on-line analysis. The substrate molecule may initially be added once and remain present while samples are collected over the desired time period. Alternatively, the substrate molecule is added in an appropriate amount at each point in time immediately prior to detecting the released moiety. When quantitative detection of the released moiety is desired, the substrate molecule must be present at a concentration representing xe2x80x9csaturationxe2x80x9d. This can also be achieved by using subsaturation concentrations if only 10-20% of the substrate molecules are cleaved, preferably at a constant xe2x80x9cturnoverxe2x80x9d rate, so as to release the detectable moiety at a constant rate over a suitable time interval.
The wide range of applications based on the present method is illustrated by the fact that the sample may be contacted with a substrate molecule either in a liquid or a solid medium. Thus, both fungal and bacterial species can be screened for enzymatic activity by using a suitable solid medium, such as e.g. soil extract agar (SEA) medium, supplemented with 4-MU-GlcNAc. The screening medium may also be supplemented with, substances having potential inducing effects as it is described in the following examples.
Contacting a sample with the substrate molecule may also lead to xe2x80x9cin situ detectionxe2x80x9d of a fungal biomass. As used herein, xe2x80x9cin situ detectionxe2x80x9d designates determination of the fungal biomass in its place of growth such as e.g. a natural habitat. There is also provided a method for determining fungal biomass present beneath the outer surfaces of the sample.
In particularly preferred embodiments of the invention, the sample is an environmental sample collected from soil, water or air. The sample can also be obtained or derived from a building element or be a sample of wood. In one particular embodiment, there is provided a method of detecting Tuberaceae and/or Cantharellus biomass in a sample collected from soil. State of the art methods currently available for determining Tuberaceae biomass requires, besides being time consuming and elaborative, the use of experimental animals, such as e.g. pigs.
In accordance with the invention, fermented products for human or animal consumption, a field crop or part hereof, a plant or part hereof, a vegetable and a fruit can be assayed for the presence of fungal biomass. Assaying grains, seeds or nuts for the presence of fungal biomass is a particularly useful application.
In other applications, a sample of a field crop, a plant, a vegetable or a fruit is assayed and the results can be evaluated in the context of monitoring and controlling the distribution and efficacy of fungicides. Harvested products are assayed, in particular during storage, to evaluate the level of fungal contamination. The harvested products can be assayed by directly contacting a sample hereof with the substrate as described above or by first preparing a suitable extract from the harvested product to be assayed.
The method is suitable for assaying a food product e.g. a heat processed food product, a food component, a feed product and a feed component. Assaying a spice, tea, cocoa or coffee is also a useful application.
Use of the provided methods for the quantitative detection of fungal biomass generates reliable results in a sensitive manner. The obtained results can be used in evaluating compliance with general health and/or safety guidelines. In some cases, such results may be generated on the basis of non-quantitative detection of fungal biomass.
As an example of the use of the method in evaluating compliance with general health and/or safety guidelines, the ecologically and/or toxicologically effects of e.g. chemicals including organic compounds having putatively undesirable effects on an environment can be assessed. Such chemicals are added to selective samples of e.g. soil comprising a fungal biomass and the effect of the chemical on the growth of the fungal species is determined. In one particularly interesting embodiment, the diffusion of fungicides into a sample of soil supplemented with a fungal biomass is evaluated in terms of monitoring the survival of the fungal biomass. This survival may be correlated to traditional fungus indicators, such as e.g. ergosterol and PLFA.
In a particular application of the method, the sample comprises a fungal biomass used in the production of a desired product in the form of a peptide, such as a polypeptide, an enzyme, an epitope, a hormone, an anti-viral protein, an anti-tumour protein and a growth factor. The fungal biomass may also be used in the production of a desired metabolite or a cellular component. The above fungal biomass may comprise at least one recombinant fungal species or a species comprising a gene and/or expression signals not naturally associated with said fungal species. The term xe2x80x9crecombinantxe2x80x9d refers to any form of genetic engineering used to produce the recombinant species. The fungal biomass may also be one wherein the production of a polypeptide, a metabolite and/or a cellular component has been reduced or eliminated, said reduction and/or elimination being achieved by traditional screening techniques or by techniques involving recombinant genetics and/or genetic engineering.
In a contemplatable aspect of the invention, means for detecting the released moiety by e.g. a fluorometer is provided. Accordingly, the invention also pertains to an apparatus for fluorometrical and/or chromogenical detection of released and detectable moieties generated in the method described above. The apparatus is suitably a portable apparatus which can be used for xe2x80x9cplatformxe2x80x9d or field tests.
In yet another contemplatable aspect of the invention, there is provided a method of determining a microbial biomass by detection of a detectable enzymatic activity present in said biomass. In particular, there is provided a putative method comprising the steps of (i) contacting a sample with a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of a selectively detectable microbial enzymatic activity, and (ii) detecting the released moiety.
A further interesting application of the present method is, the detection of fungal infections in humans and animals. Thus, it was found that the method permitted the detection of fungally derived xcex2-N-acetylhexosaminindase present in human plasma samples. However, it was also discovered that human plasma samples have an inherent hexosaminidase activity, which could be removed from the plasma e.g. by precipitation with ammonium sulphate. Such a pre-treatment did not affect the detection of the fungally derived xcex2-N-acetylhexosaminidase also present in the plasma. It is envisaged that the present method can be used generally for detection of fungi or fungally derived enzymatic activity in body samples from humans and animals.
The invention pertains, as it is mentioned above, to a product comprising (i) an agent that reacts with an enzyme that is present in substantially all fungal species and the amount or activity of which enzyme is correlated with the amount of fungal biomass present in a sample, the reaction between the enzyme and the agent resulting in a detectable signal and (ii) means for detecting said signal, as a combined system for the detection of a fungal biomass present in the sample. Thus, the agent of such a product can be selected from a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of the fungal enzyme, an immunologically active compound including a polyclonal or monoclonal antibody capable of binding to the fungal enzyme, and a nucleotide sequence that is capable of hybridizing to a DNA or RNA sequence coding for the fungal enzyme of interest.
Suitable means for detecting the signal include a spectrometer of any of the above types.